A simple procedure for the preparation of pure kinetoplast DNA network free of nuclear DNA from the kinetoplast hemoflagellate Leishmania donovani

A simple, inexpensive procedure for preparing pure kinetoplast DNA network from Leishmania donovani is described. L. donovani promastigotes were lysed by incubating with pronase in presence of sodium dodecylsulfate. Crude kinetoplast DNA networks were obtained by centrifugation of the lysate through a 20% sucrose solution. The pellet containing kinetoplast DNA was deproteinized by phenol extraction. Contaminating nuclear DNAs were removed by denaturation with alkali, neutralization, and addition of polyethylene glycol-8000 to a concentration of 10% to facilitate precipitation of kinetoplast DNA. kDNA isolated after centrifugation was deproteinized several times with phenol and finally precipitated with ethanol. The average yield by this procedure is 30-50 micrograms of kDNA per gram of wet cells. By slot-blot hybridization with a nuclear DNA probe, no nuclear DNA contamination of the kDNA networks could be detected.     

Disco-ordinate expression of the Escherichia coli gal operon after prophage lambda induction.

EcoRI endonuclease crystallizes in space group C2 with unit cell parameters $\text{a = 209}\text{A}\text{?}\text{, b = 129}\text{A}\text{?}\text{, c = 50}\text{A}\text{?}\text{and}\text{β = 98.4 °}$. Four 29,000 molecular weight subunits per asymmetric unit would give a reasonable V_m value of 2.87 ų/dalton. EcoRI endonuclease is the first protein which recognizes a specific sequence of bases in DNA to be crystallized in a form suitable for high resolution structure analysis.     

RNA-mediated restoration of mitochondrial function in cells harboring a Kearns Sayre Syndrome mutation

Mutations in mitochondrial DNA (mtDNA) generate multi-system disorders due to failure of ATP production. A cybrid containing a 1.9-kb mtDNA deletion from a patient with Kearns Sayre Syndrome is respiration-defective and grows glycolytically. When treated with a ribonucleoprotein (RNP) complex of polycistronic RNA 1 (pcRNA1) containing mtDNA-encoded genes and a multi-subunit carrier complex R8, full-length pcRNA1 was transported to mitochondria. Translation of the pcRNA1-encoded mRNAs was observed in mitochondria from RNP-treated cells. Respiration of the cybrid was rescued to approximately 90% of normal within hours, switching the cells to aerobic growth. These findings have implications for the development of effective mitochondrial gene therapy.     

Mitochondrial RNA import in Leishmania tropica: aptamers homologous to multiple tRNA domains that interact cooperatively or antagonistically at the inner membrane

A large number of cytoplasmic tRNAs are imported into the kinetoplast-mitochondrion of Leishmania by a receptor-mediated process. To identify the sequences recognized by import receptors, mitochondria were incubated with a combinatorial RNA library. Repeated cycles of amplification of the imported sequences (SELEX) resulted in rapid selection of several import aptamers containing sequence motifs present in the anticodon arm, the D arm, the V-T region, and acceptor stem of known tRNAs, confirming or suggesting the presence of import signals in these domains. As predicted, truncated derivatives of tRNA(Ile)(UAU) containing the D arm or the V-T region were imported in vitro. Four aptamers were studied in detail. All were imported in vitro as well as in transiently transfected cells, using the same pathway as tRNA, but their individual import efficiencies were different. Two types of aptamers were discernible: the A arm and D arm homologues (type I), which were efficiently transferred across the inner mitochondrial membrane, and the V-T homologues (type II), which were not. Remarkably, subnanomolar concentrations of type I RNAs stimulated the entry of type II RNAs into the matrix, whereas type II RNAs inhibited inner membrane transfer of type I RNAs. Moreover, tRNA(Tyr)(GUA) and tRNA(Ile)(UAU) interacted with one another as type I and type II, respectively. Such cooperative and antagonistic interactions may allow the use of a limited number of receptors to recognize a large number of tRNAs of variable affinity and enable the maintenance of a properly balanced tRNA pool for mitochondrial translation.     

Dynamics of changes in methanogenesis and associated microflora in a flooded alluvial soil following repeated application of dicyandiamide, a nitrification inhibitor

Influence of repeated application of the nitrification inhibitor dicyandiamide (DCD), on CH(4) production and associated microflora in a flooded alluvial soil, was investigated in a laboratory incubation study. Application of DCD at the time of soil incubation resulted in a substantial reduction in CH(4) production (31% over that of untreated control). Second repeat application of DCD, on the contrary, annulled the inhibitory effect on CH(4) production, restoring it to the level of unamended soil. Application of the third dose of DCD maintained CH(4) production almost to the same extent as that of second application. The alleviation of the initial inhibitory effect of DCD on CH(4) production was linked to the enhanced degradation of DCD following its repeated application to the flooded soil. Admittedly, abatement of the initial inhibitory effect of DCD on CH(4) production in soil repeatedly amended with DCD was also related to the inhibition of CH(4)-oxidizing bacterial population and noticeable stimulation of heterotrophic bacterial population. Results suggest that repeat application of DCD with fertilizer-N to flooded rice soils might not be effective in controlling CH(4) production under field condition.     

Bacteriophage infection is targeted to cellular poles

The poles of bacteria exhibit several specialized functions related to the mobilization of DNA and certain proteins. To monitor the infection of Escherichia coli cells by light microscopy, we developed procedures for the tagging of mature bacteriophages with quantum dots. Surprisingly, most of the infecting phages were found attached to the bacterial poles. This was true for a number of temperate and virulent phages of E. coli that use widely different receptors and for phages infecting Yersinia pseudotuberculosis and Vibrio cholerae. The infecting phages colocalized with the polar protein marker IcsA-GFP. ManY, an E. coli protein that is required for phage lambda DNA injection, was found to localize to the bacterial poles as well. Furthermore, labelling of lambda DNA during infection revealed that it is injected and replicated at the polar region of infection. The evolutionary benefits that lead to this remarkable preference for polar infections may be related to lambda's developmental decision as well as to the function of poles in the ability of bacterial cells to communicate with their environment and in gene regulation.